1. Field of the Invention
The present invention relates to improved gene transfer methods and, more particularly, methods that enable highly efficient and widespread delivery of selected nucleic acids, to solid organs such as the heart or liver as well as to other solid cell masses such as a solid tumor.
2. Background
Effective delivery of nucleic acid to cells or tissue with high levels of expression are continued goals of gene transfer technology. As a consequence of the general inability to achieve those goals to date, however, clinical use of gene transfer methods has been limited.
Thus, for example, several delivery schemes have been explored for in vivo myocardial gene transfer, but none has proven capable of modifying a majority of cardiac myocytes in a homogeneous fashion. Techniques involving injection directly into the myocardium are considered of limited use because gene expression does not extend significantly beyond the needle track. R. J. Guzman et al. Circ Res 1993; 73:1202-1207; A. Kass-Eisler Proc Natl Acad Sci 1993; 90:11498-11502. In one study, percutaneous intracoronary delivery of 1010 pfu of adenovirus caused infection in only about one-third of the myocytes in the region served by the target artery. E. Barr et al. Gene Therapy 1994, 1:51-58.
Other coronary delivery models, either in situ or ex vivo, have produced a very small percentage of infected cells spread throughout the heart. J. Muhlhauser et al. Gene Therapy 1996; 3:145-153; J. Wang et al. Transplantation 1996; 61:1726-1729. To date, no in vivo delivery system has been able to infect a majority of cells in an intact heart.
Certain gene delivery procedures also have been quite invasive and hence undesirable. For example, one report describes essentially complete loss of endothelium by mechanical or proteolytic means to enable gene transfer from blood vessels to cells positioned across interposing endothelial layers. See WO 93/00051.
Certain gene transfer applications also have been explored in other organs such as the liver. In particular, ex vivo strategies have included surgical removal of selected liver cells, genetic transfer to the cells in culture and then reimplantion of the transformed cells. See M. Grossman et al., Nat Genet 1994, 6:335-341. Such an ex vivo approach, however, suffers from a number of drawbacks including, for example, the required hepatocyte transplantation. M. A. Kay et al., Science 1993, 262:117-119; and S. E. Raper et al., Cell Transplant 1993, 2:381-400. In vivo strategies for gene transfer to the liver also have been investigated, but have suffered from low delivery efficiencies as well as low specificity to the targeted tissue. N. Ferry et al., Proc Natl Acad Sci USA 1991, 88:8377-8391; A. Lieber et al. Proc Natl Acad Sci USA 1995, 6:6230-6214; A. L. Vahrmeijer et al., Reg Cancer Treat 1995, 8:25-31. See also P. Heikkilia et al., Gene Ther 1996, 3(1):21-27.
Gene transfer has been generally unsuccessful in additional applications. For example, gene transfer therapies for treatment of cystic fibrosis have largely failed because transduction of insufficient numbers of cells.
It thus would be desirable to have improved methods and systems to effectively deliver nucleic acid to targeted cells and tissue. It would be particularly desirable to have new methods and systems for effective delivery of nucleic acids into solid organs, especially the heart, liver, lung and the like, as well as other solid cell masses such as a solid tumor.
We have now found methods and compositions that enable effective delivery of nucleic acids to desired cells, including to a solid mass of cells, particularly a solid organ such as a mammalian heart, liver, kidney, skeletal muscle, spleen or prostate, or to malignant cells such as a solid tumor. These methods and compositions enable effective gene transfer and subsequent expression of a desired gene product to a majority of cells throughout a solid cell mass, and/or gene transfer and subsequent expression of a desired gene product to a solid cell mass in a desired percentage of total cells of the mass, including up to nearly 100% of targeted cells of the mass. For example, using methods and compositions of the invention, greater than 90 percent of total cardiac myocytes showed expression of nucleic acid that was perfused for two minutes through an intact rabbit heart.
Methods and compositions of the invention preferably provide enhanced vascular permeability that enables increased nucleic acid delivery to targeted cells. While not being bound by theory, it is believed these methods and compositions of the invention induce transient permeability or interruption of endothelial layers to thereby enhance gene transfer efficiency. This is distinguished from prior approaches that significantly degraded or injured endothelial cell layers in attempts to administer nucleic acid.
Such enhanced permeability can be readily accomplished by one of several alternative approaches, or by a combination of strategies. A preferred approach provides for use of a vasculature permeability agent. As demonstrated in the Examples which follow, use of a suitable permeability agent significantly enhances transfer of administered nucleic acid to targeted cells. A permeability agent suitably may be administered through the vasculature of targeted tissue prior to administration of nucleic acid, and/or the permeability agent and exogenous nucleic acid can be administered simultaneously. Preferably, the vasculature of targeted tissue is pretreated with a permeability agent.
Preferred vasculature permeability agents include serotonin and bradykinin. Other suitable permeability agents will include platelet-activating factor (PAF), prostaglandin E1 (PGE1), histamine, vascular endothelium growth factor (VEGF), zona occludens toxin (ZOT), interleukin-2 and other plasma kinins in addition to bradykinin. Nitric oxide inhibitors, e.g. L-N-monomethyl arginine (L-NMMA) and L-N-nitro-arginine methyl ester (L-NAME), also can provide suitable results, although these agents may be less preferred than others such as serotonin and bradykinin. Other suitable agents can be readily identified, e.g. simply by testing a candidate permeability agent to determine if it enhances uptake of nucleic acid by targeted tissue relative to a control tissue sample that has not been exposed to the candidate permeability agent. A single or a combination of more than one distinct permeability agents may be administered in a particular application. In this regard, a particular application can be optimized by selection of an optimal permeability agent, or optimal xe2x80x9ccocktailxe2x80x9d of multiple permeability agents. Such optional agent(s) can be readily identified by those skilled in the art by routine procedures, e.g. testing selected permeability agents and combinations thereof in in vivo assays.
Low extracellular calcium ion concentration conditions also can be used to enhance vascular permeability. It has been found that transfer of administered nucleic acid to targeted cells is substantially enhanced under such conditions, which also is demonstrated in the Examples which follow. Low calcium concentration conditions may be readily provided, particularly by perfusing a low calcium ion concentration fluid through the vasculature of the tissue to which nucleic acid is administered. Suitable perfusate calcium ion concentrations may range from about 40 or 50 xcexcmol/L to about 500 xcexcmol/L, more preferably from about 50 xcexcmol/L to about 200 xcexcmol/L. A perfusate calcium concentration of about 50 xcexcmol/L is particularly preferred. Calcium ion (e.g. Ca2+) concentration also can be lowered through use of a suitable buffer such as a chelating agent, e.g. ethylenebis(oxyethylenenitrilo)tetracetic acid (EGTA), ethylenediaminetetracetic acid (EDTA), or 1,2-bis-(2-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (BAPTA).
Additionally, while a low calcium ion concentration can enhance nucleic acid uptake, it is also important that a minimal calcium concentration be maintained during the gene transfer protocol, at least in many or some applications. If calcium-free or essentially calcium-free conditions (e.g. perfusate calcium ion concentration of about 10-20 xcexcmol/L or less) are employed, cell calcium channel selectivity may be destroyed which can result in cell death upon return to physiological calcium levels, particularly in the case of administration to myocytes.
We have also found that combined use of a vasculature permeability agent and low calcium ion concentration conditions appears to provide synergistic results with higher gene transfer efficiency than that provided with either use of a permeability agent without a lowered calcium concentration, or low calcium concentration conditions in the absence of a permeability agent. Thus, as discussed above and demonstrated in the Examples which follow, greater than 90 percent of rabbit total cardiac myocytes showed expression of nucleic acid that was perfused for two minutes through an intact rabbit heart under low calcium ion concentration conditions and treatment with a permeability agent.
We also have found that certain administration conditions in addition to those discussed above also will impact efficiency of nucleic acid uptake by targeted tissue. In particular, concentration, amount and exposure time of the administered nucleic acid and temperature of the targeted tissue all can effect the rate of gene transfer. Flow rate and perfusion pressure also can effect the rate of gene transfer.
More specifically, in a perfusion administration protocol, the rate of gene transfer increases with increase in concentration, total amount and exposure time of the administered nucleic acid in a perfusate. High concentration of nucleic acid in a perfusate especially can increase gene transfer rate. Preferred perfusate concentrations can vary with a number of factors including the particular organ or cell mass being treated, the particular cloning vehicle being administered and the like. However, in general, preferred concentrations of a viral vector in an administered perfusate are about 1xc3x97108 pfu/ml or greater, more preferably a concentration of about 5xc3x97108 pfu/ml or greater. Administered perfusate also preferably may be recirculated and readministered to a subject, e.g. to limit the total viral burden introduced into the target, or if the administered agent is in short supply. However, effective gene transfer can be achieved without recirculation, particularly if other delivery parameters are optimized. Increases in either flow rate of perfusate pressure generally will increase gene transfer efficiency, although for clinical safety it can be desirable to limit both perfusate pressure and flow rate.
The rate of gene transfer also decreases with decreased temperature of targeted tissue, particularly where the tissue is below about 20xc2x0 C. Delivery of the nucleic acid near body temperature of the subject is preferred, e.g. where the nucleic acid is administered at a temperature of from about 28-45xc2x0 C., more preferably from about 34-40xc2x0 C. However, gene transfer can be achieved over wide temperature ranges, e.g. at about 4xc2x0 C. Clinical circumstances may require lower temperatures, e.g. with gene transfer during cardiac surgery.
Nucleic acid administered in accordance with the invention can express a desired therapeutic agent, or may inhibit expression or function of an endogenous gene of a subject. Nucleic acid also may be administered for diagnostic purposes, for example to express a marker protein. In addition to such therapeutic and diagnostic methods, methods and compositions of the invention also may be employed to examine the effect of a heterologous gene on an intact organ such as a subject""s heart, to create animal models of disease and to provide mechanistic information regarding various disease states.
In a preferred aspect, the invention includes methods for xenotransplantation. Thus, for example, cells of xenogeneic tissue, particularly cells of a xenogeneic solid cells mass, can be administered exogenous nucleic acid under enhanced vascular permeability as described herein. Those cells containing exogenous nucleic acid then may be transplanted into a subject. More particularly, the exogenous nucleic acid can be administered in vivo or ex vivo to donors cells or organs, e.g. a xenogeneic heart, liver, spleen and the like, in accordance with the invention and the donor cells or organ can be transplanted to a selected host, e.g. a mammal, particularly a primate such as a human. Suitable donor organs may be obtained from e.g. another primate, or a swine, particularly a pig. A variety of exogenous nucleic acids can be administered to the donor cells. For instance, nucleic acids can be administered that will express a gene product that can promote a desired phenotypic characteristic. Exemplary gene products include those which can reduce immune system recognition of the xenotransplanted cells.
In another aspect, the invention includes vasculature permeability and gene transfer solutions useful in the methods of the invention. Such solutions in general will be formulated to provide enhanced permeability to treated tissue upon administration of nucleic acid to such layers. Thus, suitable permeability agents will include one or more permeability agents as disclosed herein, or otherwise may be formulated to enhance permeability such as a low calcium ion concentration solution as described herein. A solution of the invention also may contain one or more therapeutic agents, e.g. one or more exogenous nucleic acids (e.g. one or more recombinant adenoviruses) to be administered to a subject, or other pharmaceutical agent such as nitroglycerine to control vasospasms and the like to a solution that will be administered to a heart. A permeability or gene transfer solution may suitably contain nucleic acid in a form for administration e.g. in a suitable cloning vehicle such as a viral vector dissolved in desired pharmaceutically acceptable carrier e.g. Krebs solution or other buffered solution. A permeability or gene transfer solution of the invention preferably will be pharmaceutically acceptable, e.g. sterile and otherwise suitable for administration to a subject. Typically a vasculature permeability or gene transfer solution will be stored in a sealed (preferably, hermetically sealed) container prior to use. A permeability or gene transfer solution preferably will contain active ingredients (i.e., permeability agent, calcium ion concentration, nucleic acid) in optimal dosage quantities. Solutions of the invention may suitably have other agents such as various buffers, sugars, salts and the like.
A wide variety of cells may be treated in accordance with the invention. Suitable cells for administration include those that have a distinct circulation, or circulation that can be isolated in some manner. Thus, for example, organs and other cell masses are suitable for administration in accordance with the invention including e.g. heart, lung, liver, kidney, prostrate, testes, ovaries, skeletal muscle, kidneys, brain, spleen and solid tumors. Exemplary tumors that can be treated in accordance with the invention include e.g. cancers of the lung, prostate, liver, brain, testes or ovaries. Cells treated in accordance with the invention may be in either a healthy or diseased state. A wide variety of subjects also may be treated in accordance with the invention. Typical subjects include mammals, particularly primates, especially humans.
Other aspects of the invention are discussed below.